PB1762: A NEW MONOCLONAL ANTIBODY PROVIDES INSIGHTS ON NPM1 MUTANT SUBCELLULAR EXPRESSION, INTRACLONAL CELL DIFFERENTIATION AND MRD IN NPM1-MUTATED AML
Authors: Brunangelo Falini, Maria Paola Martelli, Riccardo Rossi, Fabio Facchetti, Stefania Zini, Erica Borlenghi, Elena Sabbatini, Lorenzo Brunetti, Daniele Sorcini, Vincenzo Perriello, Enrico Tiacci, Bjorn Gjertsen, Vibeke Andresen
Journal: HemaSphere (2023)

Topic: 3. Acute myeloid leukemia - Biology & Translational Research Background:NPM1-mutated AML is a distinct WHO and ICC entity characterized by unique clinical and molecular features, including the aberrant cytoplasmic dislocation of the NPM1 mutant protein that is critical for leukemogenesis and can be tracked in tissue sections by immunohistochemistry. Aims: We generated a monoclonal antibody (mAb) specifically recognizing the mutant but not wild-type NPM1 protein that is suitable for immunostaining of bone marrow (BM) paraffin sections to address the following issues: i) Expression of NPM1 mutant protein in human normal and neoplastic tissues; ii) Subcellular expression of NPM1 mutant in NPM1-mutated AML; iii) Evaluation of NPM1-mutant expression and clonality during monocytic differentiation; and iv) Assessment of minimal measurable disease (MRD). Methods: Immunostainings with the specific mAb on paraffin sections and molecular analyses were performed according to standard procedures Results: This mAb was tumor specific since it strongly immunostained leukemic cells in 128/128 NPM1-mutated AMLs (including 32 myeloid sarcomas) whilst was unreactive with 93 AMLs with a genotype other than NPM1 mutated and 150 tumors of different variety. NPM1 mutant positivity was mainly found in the cytoplasm of leukemic cells but about 30% of cases showed also nuclear positivity. This finding was especially evident in the leukemic proerythroblasts. This is in keeping with the recent observation that the NPM1 mutant can interact with XPO1 bound to chromatin to exert its leukemogenic activity. About 40% of NPM1-mutated AML shows monocytic maturation but how the NPM1 mutant expression correlates with differentiation and whether cells in the terminal phase of monocytic differentiation belong to the leukemic clone remains unknown. To address this issue, we immunostained BM biopsies from 45 NPM1-mutated AML cases of M4-M5 FAB subtype. In all samples, the NPM1 mutant was variably expressed, the most mature leukemic cells being negative or only weakly positive. Because the VAF of NPM1 mutations in these cases was frequently 40-50%, this provides an indirect evidence that (although the mutation is present) the NPM1 mutant protein is lost during monocytic differentiation. Notably, these samples retained the nuclear positivity with a mAb specific for the wild-type NPM1, suggesting that the NPM1 mutant is undergoing degradation more quickly than wild-type NPM1. Because the NPM1 mutant protein is not expressed in cells with the strongest monocytic maturation (including starry sky macrophages), this probe cannot be used to track clonality. Therefore, we focused our analysis on NPM1-mutated AMLs co-mutated for IDH1 R132H. Staining with a specific anti-IDH1 R132H mAb revealed strong positivity of the NPM1-mutant negative mature macrophages for the IDH1 mutant (Fig. 1A), clearly demonstrating for the first time that these cells belong to the leukemic clone. To explore whether the mAb was suitable for monitoring MRD, we immunostained 13 normal BM biopsies and CD34+ purified BM hematopoietic cells with the anti-NPM1 mAb but no reactivity was observed. Conversely the mAb detected even rare NPM1 mutant-expressing cells in patients in molecular relapse (Fig. 1B). Correlation with molecular studies is ongoing. Conclusion: We demonstrate that: i) the NPM1 cytoplasmic mutant can be expressed in the nucleus and is downregulated during monocytic differentiation; ii) cells in the terminal phase of monocytic differentiation may belong to the leukemic clone; and iii) the anti-NPM1 mutant mAb is tumor specific and can be used for monitoring MRD.Keywords: AML, Monocyte, Immunohistochemistry